Vol. 8(3), pp. 130-139, March 2014 DOI: 10.5897/AJFS08.059 ISSN 1996-0794 Copyright © 2014 African Journal of Food Science Author(s) retain the copyright of this article http://www.academicjournals.org/AJFS

Full Length Research Paper

Biogenic amines and microbiological profile of egyptian

Khaled Meghawry El-Zahar

Food Science Department, Faculty of Agriculture, Zagazig University, 44511 Zagazig, Egypt.

Received 14 December, 2013; Accepted 17 February, 2014

Cheeses are among those high-protein-containing foodstuffs in which enzymatic and microbial activities cause the formation of biogenic amines (BAs) from amino acids decarboxylation. Most of the methods for amine determination in these products involve acid extraction followed by a liquid-liquid purification step to selectively separate amines and amino acids. This study aimed to describe the development of biogenic amines in Egyptian cheeses during ripening and storage regimes. Biogenic amines content in , Ras and Blue cheeses were 270-1300, 340-980 and 210-700 mg/kg, respectively. The dominant biogenic amines were different. This work confirms that the main biological feature influencing amines formation is the extent of growth of microorganisms, like Enterococci, characterized by decarboxylase activity. It is important to note that the presence of biogenic amines due to the activities of these microorganisms is maintained within safe levels. In Egypt, reports dealing with the Egyptian cheeses (Mish, Ras and Blue) are scanty. So, the present work was carried out to fill the gap in our knowledge on its microbiological and biochemical features, focusing on hygiene and consumer health aspects.

Key words: Biogenic amines, food safety, proteolysis, ripening, Enterococci spp.

INTRODUCTION

Milk and milk products are very important in human which they are responsible for many essential functions. nutrition and, among them; is considered a good They can be naturally present in many foods such as source of proteins, vitamins and minerals. However, fruits and vegetables, meat, fish, chocolate and milk, but cheese is one of the most fermented foods commonly they can also be produced in high amounts by associated with biogenic amines (BAs) contamination. microorganisms through the activity of amino acid These compounds are basic nitrogenous compounds decarboxylases (Ten Brink et al., 1990). Excessive formed by series of microorganisms, mainly by consumption of these amines can be of health concern decarboxylation of amino acids or “in vivo” also by de- because when there is are no equilibrate assumption in amination and trans-amination of aldehydes and ketones human organism, can generate different degrees of (Loizzo et al., 2012, 2013). Biogenic amines are diseases determined by their action on nervous, gastric compounds commonly present in living organisms in and intestinal systems and blood pressure (Suzzi and

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Gardini, 2003). Biogenic amines are low molecular weight (Wöhrl et al., 2004). nitrogenous bases, they were found in fermented foods The ability of microorganisms to decarboxylate amino and cheese (Mohamed et al., 2013). acid is highly variable. Due to strain-specific, it is impor- Also, biogenic amines are low-molecular nitrogenous tant to count decarboxylase-positive microorganisms to compounds that are formed in foodstuffs mainly by estimate; the risk of BAs food content and to prevent BAs microbial decarboxylation of the precursor amino acids accumulation in food products. Presence and accumula- (Alberto et al., 2002). The importance of observing BAs tion of BAs depends on many factors such as presence content lies in potential toxicity to human, mainly when of specific bacteria (Enterococci, Micrococci, the concentration is up to 100 mg/kg (or up to 100 mg/L). Enterobacteriaceae and Lactobacilli) and enzymes, Thus, the presence of BAs significantly influences the availability of free amino acids, presence of suitable food quality and safety (Smit et al., 2005). cofactors, that is, pH level, water activity, temperature The presence of relevant amounts of BAs in cheeses and salt content, type of cheese, ripening and storage has been documented (Martuscelli et al., 2005; Kung et period (Galgano et al., 2001). Some controversial results al., 2007; Pintado et al., 2008; Ladero et al., 2009; have been reported on the contribution of Enterococci sp. Mercogliano et al., 2010). In cheeses, BAs formation is in BAs production in cheeses, and in particular in hista- caused by curdling and cheese decarboxylase-positive mine (Sumner and Taylor, 1989). Enterococci have a microorganisms. Histamine (HIS), tyramine (TYR), long history of use as artisanal cultures for preparation of putrescine (PTR), cadaverine (CAD), spermidine (SPD), various types of cheeses (Izquierdo et al., 2009), they are spermine (SPR), tryptamine (T), and β-phenylethylamine sometimes associated with pathogenicity (Khan et al., (PE) are frequently found in these products. Cheese is 2010), can cause endocarditic, bacteraemia, and several one of the fermented foods most commonly associated infections, as well as multiple antibiotic resistances with BAs poisoning; mainly HIS, TYR, PTR and CAD. (Kayser, 2003). Although, the chemical composition and Indeed, the term ‘‘cheese reaction” to refer to it (Ten microbiological quality of cheeses in Egyptian markets Brink et al., 1990). Tyramine and histamine are the most have been studied extensively, little data is available on abundant and frequent BAs in cheese (Fernández et al., the occurrence of biogenic amines in Egyptian cheeses. 2007). Consumption of food containing high levels of BAs Therefore, this survey was undertaken to determine the is considered undesirable since it can be associated with presence of BAs in commercially available cheeses several toxicological problems such as respiratory during ripening and storage, also to make an assessment distress, headache, hyper- or hypo-tension or allergies of the health hazard arising from the consumption of (Ladero et al., 2010). These problems are especially these products especially by susceptible individuals. severe in consumers with low levels of the enzymes Other studies such as Lorencová et al. (2012) and involved in the detoxification system (mono and di-amine Buňková et al. (2010) deal with selection and study of oxidases), either by genetic disorders (Caston et al., microorganism (such as lactic acid bacteria), which are 2002) or medical treatments (Halász et al., 1994). The major producer of biogenic amine. However, these works content of biogenic amines and polyamines significantly explore the particular biogenic amine production in differed according to the technology of ripening. The growth medium, where the concentration of biogenic cheeses unwashed during ripening had much higher amine could be biased optimal environment for the contents of all observed amines and polyamines in bacteria metabolisms. Moreover, some strains of the comparison with the washed-rind cheeses. The mean starter lactic acid bacteria (such as Lactococcus lactis content of putrescine, cadaverine and tyramine exceeded subsp. lactis) have decarboxylase activity that was 100 mg/kg in unwashed-rind cheeses, while the other observed in model environment of growth broth. amines occurred at lower levels. The content of all Behaviour of these strains has not been investigated in detected amines was very low in washed-rind cheeses; real system of the cheese and can be different in no tryptamine, phenylethylamine and histamine were comparison with condition in growth broth. found. The effect of storage on the amine formation was The objective of our pilot study was to compare the not confirmed (Samková et al., 2013). BAs content and other selected parameters in Egyptian Physiologically, histamine is one of the most effective cheese and to review hypothesis that the BAs content BAs; it has vasoactive and psychoactive effects (Repka- developed during the ripening and storage period is Ramirez and Baraniuk, 2002). Moreover, it is the main related to the presence of decarboxylase positive strain BAs involved in food poisoning and it is limited in some of Enterococci sp. foodstuffs by law. At non-toxic doses, food borne histamine can cause intolerance symptoms such as MATERIALS AND METHODS diarrhoea, hypotension, headache, pruritus and flushes. Just 75 mg of histamine, a quantity commonly present in Cheese samples some meals, can induce symptoms in the majority of healthy persons with no history of histamine intolerance A total 85, 49 and 44 of Mish, Ras and Blue cheeses samples were

132 Afr. J. Food Sci.

purchased from different Egyptian retail markets and small scale Microbiological analysis factories. The samples collected were 6-48 months old. They were kept in sterile plastic bags and transported to the laboratory of Food For each cheese sample, 10 g was weighed and dispersed asep- Science Department, Zagazig University (Egypt), then stored at 4 tically in 90 mL of citrate buffer (2%,w/v) and homogenized in a ±1°C until analyzed. sterile polyethylene bag using a Stomacher (Seward Laboratory Blender Stomacher 400 Lab Blender UK) for 1.5 min. Serial dilu- tions were made in 0.1% sterile peptone water and all determina- Chemical analysis tions were made in triplicates (Messer et al., 1985). The enumera- tion of total mesophilic bacteria (Plate Count Agar, Merck, Cheeses were analyzed in triplicates for moisture by the oven Germany) at 30°C/48 h, total coliform groups (Violet Red Bile Agar, drying method at 102°C (IDF, 1993), salt by titration with AgNO3, Merck, Germany) at 37°C/48 h, yeasts and moulds (Potato Dex- and fat by Gerber method (AOAC, 2002). For pH measurement, trose Agar, Merck, Germany) at 21°C/7 days, Lactobacilli (MRS grated cheese (10 g) was macerated with 10 mL of distilled water agar, Merck, Germany), Lactococcus sp. (M17 agar, Merck, Germany) and the pH of the resultant slurry was measured using a digital pH and Enterococci (Azide Dextrose agar, Merck, Germany) at meter (pH 211, Hanna Instruments, Vila do Conde, Portugal). 28°C/48 h (Frank et al., 1993) were performed. Titratable acidity was determined as g lactic acid/100 g cheese is using the method of AOAC (2002). Total volatile fatty acids and total nitrogen (TN) were determined using the methods of AOAC Statistical analysis (2002). All analyses were carried out in triplicates.

The effect of time of ripening on all parameters of proteolysis and

on total FAA and BAs content of the cheese was assessed by Assessment of proteolysis analysis of variance (ANOVA) using the SPSS 10.0 for Windows

software (Liu et al., 2003). Water-soluble nitrogen fraction (WSN) of cheese was prepared according to Kuchroo and Fox (1982) and a cheese to water ratio of 1:5 was used. 12% trichloroacetic acid soluble nitrogen-fraction (TCA-SN, that is, NPN) was obtained by mixing equal volumes of RESULTS AND DISCUSSION water-soluble fraction and 24% (w/w) TCA solution, followed by filtration through a white ribbon filter paper (Schleicher and Schuell, Evaluation of physico-chemical parameters Dassel, Germany). The nitrogen content of both fractions WSN and TCA-SN, respectively, was determined by Kjeldahl method (AOAC, 2002) and expressed as percentage of TN. The chemical compositions of Egyptian cheeses are presented in Table 1. The total solids content of cheese samples varied from 30.5 to 46.5, 47.2 to 58.3 and 41.2 Free amino acids and biogenic amines to 48.8% in Mish, Ras and Blue cheeses, respectively. A significant variation of fat content was observed, 17.8- Free amino acids (FAA) and BAs were assayed according to the method of Krause et al. (1995), modified by Pinho et al. (2001). In 30.4; 33.8-48.3 and 25.3-38.4% in Mish, Ras and Blue brief, a 4 g cheese sample was suspended in 15 mL of 0.2 M cheeses, respectively. The salt content of the cheese aqueous perchloric acid; the mixture was homogenized in an Ultra samples fell within the range, 6.1-10.5; 5.6-6.8 and 4.5- Turrax blender (Sotel, Warsawa, Poland) for 2 min, then kept in an 5.7% in Mish, Ras and Blue cheeses, respectively. ultrasonic bath (Heraeus, Osterode, Germany) for 30 min, and Whereas, the pH of cheeses ranged from 4.2 to 5.3; 4.5- finally centrifuged at 4000 xg for 20min. Derivatization was carried 5.2 and 4.9-5.8 respectively, which agrees with those out via dansyl chloride, at 70°C per 15 min. The reaction was quenched by placing the vials in an ice bath for 5 min. High reported for good quality Egyptian cheeses (Kebary et al., performance liquid chromatography (HPLC, Waters 600) was used 1999; Ibrahim and Amer, 2010). Total nitrogen content in to dansylamines determination. The system was equipped with cheese samples was slightly higher in Ras cheese as delivery system, reverse phase CI8 Nucleosil column 250 x 4 mm, compared to Mish and blue cheeses. Whilst, the water 10 µm packing (Macherey - Naggl). The detection was performed soluble nitrogen was lowest in as compared using U.V detector (Waters 486) at wavelength of 254 nm using linear program of 25 min period and 1 ml/min constant solvent flow to other cheeses (Table 1). The WSN/TN ratio showed rate. Data were integrated and recorded using a Millennium differences in the degree of ripening of the component Chromatography (Waters, Milford MA 01757). Elution was carried cheeses. NPN represented more than 50% of the WSN out at a flow rate of 1 mL/ min, using a volumetric gradient of of the tested cheeses; this may have originated from the solution A, 9 mM aqueous sodium dihydrogenophosphate, 4% (w/v) component cheeses. WSN and NPN have been classi- dimethyl formamide and 0.1% (w/v) triethylamine (adjusted to pH cally used as a measure of the extent of secondary pro- 6.55 with phosphoric acid), and solution B, 80% (v/v) aqueous acetonitrile. Detection was performed by measuring absorbance at teolysis, that is, formation of small sized peptides (2–20 436 nm. Quantification was carried out based on a mixture of amino residues) and free amino acids (Furtado and Partridge, acid standards: aspartic acid, glutamic acid, serine, threonine, 1988). Total volatile fatty acids showed a significant va- glycine, alanine, arginine, proline, valine, methionine, isoleucine, riation among the tested cheeses, 33.5-55.4; 62.7-92.7 leucine, lysine, histidine, tyrosine, cystine, tryptophan and and 45.6-74.5 as 0.1 N NaOH/100 g, in Mish, Ras and phenylalanine; and biogenic amine standards: ornithine, tryptamine, Blue cheese, respectively. These variations indicated phenyl ethylamine, putrescine, cadaverine, histamine, tyramine and spermine (Sigma Chemical). All determinations were performed in large differences in quality and degree of ripening in triplicates (Figure 1). Egyptian cheeses. Production of BAs has frequently been

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Figure 1. Chromatograms present the areas of biogenic amine standard solution derivatized with incubation at 40°C for 40 min.

Table 1. Chemical composition and proteolysis indices of samples ripened/stored at different periods.

Physico-chemical parameter Proteolysis indices Ripening/storage Total Solid Fat in solid Protein in Salt in Total Non-protein Water soluble Cheese type period (mon) Acidity * TVFA** (%) (%) solid (%) pH solid (%) nitrogen (%) nitrogen (%) nitrogen (%) 6 30.5±2.0d 17.8±2.5d 20.1±1.07a 3.31±0.71a 4.2±0.1d 6.1±1.5b 3.15±0.98a 0.22±0.4b 0.29±0.5d 33.5±2.2c

12 35.6±2.1c 20.8±2.2cd 18.5±1.15b 3.01±0.65b 4.4±0.1cd 7.2±1.6ab 2.91±0.78ab 0.31±0.6a 0.34±0.6cd 40.5±2.5b

24 37.0±1.9b 22.3±3.2bc 17.23±1.01b 2.8±0.52b 4.5±0.2c 8.3±2.4ab 2.71±0.73bc 0.33±0.6a 0.41±0.4bc 47.9±2.9a

36 39.4±2.5b 25.6±5.2b 14.35±0.8c 2.5±0.53c 4.9±0.2b 9.2±2.4ab 2.24±0.56cd 0.36±0.7a 0.49±0.4b 52.3±3.1a Mish 48 46.5±2.7a 30.4±4.5a 13.33±0.5c 2.2±0.44c 5.3±0.2a 10.5±2.9a 2.19±0.57d 0.39±0.7a 0.55±0.3a 55.4±3.2a 6 47.2±2.9d 33.8±1.5d 24.88±1.8a 2.2 ±0.43a 4.5±0.2b 5.6±1.4b 3.91±1.29ab 0.19±0.1ns 0.31±0.6ns 62.7±3.5d

9 50.01±3.1c 37.8±2.0c 24.44±1.3b 2.1±0.42ab 4.6±0.1b 6.7±1.5ab 3.81±1.17a 0.20±0.2ns 0.32±0.5ns 74.0±3.8c

12 52.5±3.6b 42.5±2.4b 23.6±1.4b 2.0±0.34bc 4.8±0.2b 7.3±1.7a 3.71±1.35b 0.21±0.2ns 0.33±0.5ns 85.8±4.3b Ras 24 58.3±3.8a 48.3±3.1a 21.05±1.0c 1.85±0.23c 5.2±0.1a 6.8±1.9a 3.31±1.42ns 0.25±0.4ns 0.39±0.7ns 92.7±4.5a 6 41.2±2.2c 25.3±2.1c 22.64±1.2a 1.9±0.22b 4.9±0.3b 4.5±1.2b 3.55±1.47ns 0.20±0.2b 0.25±0.3b 45.6±2.7c Blue- 9 44.5±2.4b 32.1±2.9b 21.69±1.1b 1.7±0.22c 5.1±0.5b 4.8±1.5ab 3.2±1.36ns 0.21±0.3ab 0.24±0.4b 66.6±3.7b viened 12 48.8±2.8a 38.4±3.6a 20.41±1.1c 2.8±0.34a 5.8±0.2a 5.7±1.7a 3.4±1.38ns 0.24±0.4a 0.34±0.5a 74.5±3.9a

Averaged data of analyzed cheese samples in triplicate; Mean value ± a standard deviation * Acidity is expressed as lactic acid. ** Total volatile fatty acids is expressed as 0.1 N NaOH/100 g cheese.

134 Afr. J. Food Sci.

Table 2. Levels of biogenic amines (mg/100 g cheese) of Egyptian cheese samples ripened/stored at different periods.

Cheese Ripening/storage Biogenic amines (mg/100 g) type period (month) TYR T PTR HIS CAD SPD SPR PE Total 6 6±0.62e 4±0.51d 4±0.45d 9±0.69e 3±0.3e 1±0.05d Ndc Ndd 27±2.2 12 12±1.5d 10±0.85c 10±0.85c 14±0.98d 10±0.5d 1±0.11d Ndc Ndd 57±3.2 Mish 24 14±0.42c 17±1.54b 18±1.5b 27±2.4c 18±1.72c 2±0.21c 1±0.08b 4±0.53c 101±4.3 36 15±0.46b 21±1.79a 19±1.7ab 29±2.3b 20±2.1b 3±0.42b 1±0.09b 7±0.64b 115±4.6 48 19±1.55a 22±2.15a 20±2.16a 31±2.5a 22±1.97a 4±0.63a 2±0.16a 12±1.15d 132±5.3

6 3±0.28d 10±0.93b 6±0.42d 12±0.96d Ndd 1±0.08b Ndb 3±0.19b 34±2.6 9 4±0.54c 11±1.05c 8±0.62c 14±0.91c 8±0.71c 1±0.11b Ndb 3±0.21b 49±2.8 Ras 12 5±0.61b 13±1.13b 13±0.54b 23±2.05b 13±1.26b Ndc 1±0.08a 5±0.41a 73±3.7 24 14±1.07a 20±1.86a 16±1.25a 26±2.4a 20±2.1a 2±0.21a Ndb Ndc 98±4.1

6 Ndc 11±0.45c 1±0.15c 4±0.34c 4±0.22c Ndb Ndb 1±0.11c 21±2.1 Blue- 9 1±0.1b 15±0.87b 2±0.31b 9±0.94b 7±0.65b Ndb Ndb 2±0.25b 36±2.6 viened 12 8±0.72a 17±1.26a 9±0.93a 14±1.26a 11±1.13a 3±0.41a 2±0.17a 6±0.62a 70±3.5

Averaged data of analyzed cheese samples in triplicates, nd: not detected.

referred to as the proteolytic activity of microorganisms enforced. present in cheese during manufacture and ripening. Even if no significant differences were observed in the Increases in the non-protein nitrogen fractions (WSN and final amounts of BAs in Blue and Ras cheeses, the NPN) often means level increase of free amino acids, dynamics of accumulation were not the same. Overall, which are precursors of BAs. histamine was the most prevalent amine, being found in all analyzed cheese samples. It was followed by T (98%), PTR (97%), CAD (95%), TYR (89%), SPD (73%), PE Amino acids and BAs (72%) and SPR (37%). In spite of being the most frequently detected amine, SPR was present at low BAs content of cheese can be extremely variable and levels, below 2.5 mg/ 100 g cheese. Spermidine and PE depends on the type of cheese, the ripening time, the were also detected at low levels (below 4 and 12mg/100 manufacturing process and the microorganisms present g cheese, respectively). However, HIS, CAD, T, PTR and (Ordonez et al., 1997). The Egyptian cheeses (Mish, Ras TYR were detected at levels up to 30, 21, 20, 19 and 18 and Blue) examined confirmed this variability in the total mg/ 100 g, respectively. HIS was the most prevalent content of BAs ranging from 21.0 to 130.0 mg/100 g amine, it was found in all analyzed cheese samples cheese (Table 2 and Figure 2). There are significant (Table 2). Higher means levels were detected for Mish, differences among contents of the eight BAs assayed. Ras and Blue cheeses (9-31, 12-26 and 4-14 mg/ 100 g, Only the Mish cheese contained more than 100 mg/100 g respectively). HIS levels capable of causing histamine cheese of the total BAs, as affected by increasing the poisoning were detected in all cheese samples. However, storage period. According to Taylor (1985), the threshold taking into account the concomitant presence of of risk is 100 mg/kg total amines of cheese, if ingestion is polyamines, it is likely that a higher percentage of cheese associated with such potentiating co-factors as amine samples could cause HIS poisoning. TYR was present in oxidase-inhibiting drugs or alcohol, or else if there are 100% of Mish and Ras cheeses and in 60% of Blue pre-existing gastrointestinal diseases (Stratton et al. cheese. Mish (53%) and Ras cheese (18%) contained 1991). Production of BAs in cheese has often been TYR at levels capable of causing hypertensive crisis associated with non-starter lactic acid bacteria and (Komprda et al., 2008). Overall, T was detected Enterobacteriaceae (Joosten and Northolt, 1987), so it sporadically, at lower amounts as compared to HIS. may be a toxicological risk associated with consumption Similar results were observed by Chang et al. (1985). of raw milk cheese, especially by sensitive individuals. Higher means levels of tryptamine were observed in Mish Spanjer and van Roode (1991) suggested that the total and Ras cheeses. The toxic threshold of tryptamine is not concentration of tyramine, histamine, putrescine and known (Joosten, 1988). PE, another amine of health cadaverine in cheese should not exceed 900 mg/kg DW- significance was detected 100% in Blue, 82% in Ras and 1, but no upper limit for BAs in cheese has been legally 53% in Mish cheese. The prevalence of this amine was

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Figure 2. Chromatograms presenting the concentration of histamine, tryptamine, putrescine, tyramine, phenyl ethylamine and cadaverine in Ras cheese.

high, however the levels detected were low (≤12 mg/100 Several analyzed samples did not meet the standard of g), below its toxic threshold. The rate of CAD and T identity and quality established by Egyptian legislation. accumulation were similar for the traditional Mish and With regard to quality parameters, pH, moisture and fat Ras cheeses and that described for the total BAs forma- content and acidity correlated significantly (P≤ 0.05) with tion. The accumulation of amines increased remarkably formation and accumulation of some BAs. These results later in the ripening and storage periods. The rate of PE suggest that, among quality parameters evaluated, acidity accumulation was similar for Ras and Blue cheeses, influenced amine formation in several cheese types. whereas HIS and T values were higher in the Ras cheese These results are supported by the theory that the forma- and reached concentrations of 12 - 26 mg/100 g and 10- tion of BAs is a protective mechanism of bacteria against 20 mg/100 g, respectively. acidic environments (Maijala, 1994). The presence of Similar results were obtained with heat treatment or micro-organisms with high decarboxylase activity has bactofugation of the milk used for emmental production been reported as the main factor for BAs production in and had little effect on the TYR content (Krause et al., cheese. Moreover, some strains have proteolytic activity, 1997). The rates of SPR and SPD accumulation were which can affect the accumulation of BAs in cheese similar for Mish and Blue cheeses, whereas T and HIS (Galgano et al., 2001). For the production of amines, the were higher in the Mish cheeses and reached concen- enzymatic activity of proteases derived from micro-orga- trations of 4-22 and 9-31 mg/100 g, respectively. Accor- nisms, or from another origin, is important from a qualita- ding to Halász et al. (1994), Gouda cheese along with tive point of view, that is, in relation to the type of amino Swiss and Cheddar cheeses, which contain high levels of acids provided to the amino acid decarboxylating micro- BAs are the most frequently incriminated cheese in hista- flora. The bacteriological composition of milk could be mine poisoning episodes. critical to define the amine profile in cheese; therefore, Amino acid levels in cheeses types were extremely large amounts of amines in cheese could indicate unsui- variable (Table 3). This fact was attributed to an accele- tability from a hygienic point of view, and the milk used rated amino acid release at the manufacturing day, when for cheese making. Moreover, the results emphasize the cheeses were incubated at temperatures favorable for necessity of controlling the indigenous bacterial popula- microorganism development and activity (Bütikofer and tion responsible for high production of BAs and the use of Fuchs, 1997). Any food with free amino acids, especially competitive adjunct cultures is suggested. tyrosine and phenylalanine, are subject to BAs formation if poor sanitation and low quality foods are used or if the Microbiological evaluation food was subjected to temperature abuse or extended storage time (Schirone et al., 2011). Microbiological analyses of the Egyptian cheeses were High variability was observed in pH, acidity, moisture examined throughout ripening/storage period (Table 4). and fat contents of the different analyzed cheese types. Lactic acid bacteria did not show any substantial change

136 Afr. J. Food Sci.

Table 3. Total amount of free amino acids content (mg/g dry weight of cheese) of Egyptian cheese ripened/stored at different periods.

Mish cheese Ras cheese Blue cheese Amino acid Ripening/storage period (mon) 6 12 24 36 48 6 9 12 24 6 9 12 Threonine 1.38±0.1 1.36±0.13 1.38±0.13 1.20±0.11 1.18±0.1 1.18±0.1 1.32±0.1 1.38±0.1 1.28±0.1 1.28±0.1 1.18±0.1 1.12±0.1 Serine 2.26±0.2 2.09±0.23 2.06±0.23 1.96±0.23 1.70±0.2 1.62±0.2 1.88±0.2 1.88±0.3 1.81±0.3 1.79±0.2 1.72±0.2 1.44±0.1 Glutamic 8.52±1.3 8.21±1.2 8.19±1.2 6.50±1.1 5.76±1.2 9.96±1.6 6.63±1.1 5.54±0.9 5.46±1 8.66±1.3 8.60±1.3 5.34±1 Proline 3.34±0.3 3.62±0.34 3.6±0.34 2.86±0.12 2.80±0.2 2.68±0.24 2.60±0.3 3.04±0.3 3.00±0.3 2.75±0.3 2.70±0.3 2.44±0.3 Glycine 0.92±0.1 0.79±0.11 0.77±0.11 0.80±0.1 0.86±0.09 0.92±0.11 0.80±0.1 0.72±0.08 0.71±0.08 0.85±0.1 0.82±0.1 0.74±0.08 Alanine 1.56±0.1 1.55±0.19 1.54±0.19 1.34±0.13 1.40±0.1 1.14±0.13 1.44±0.1 1.38±0.1 1.31±0.1 1.42±0.1 1.40±0.1 1.38±0.1 Cysteine 0.18±0.03 0.18±0.03 0.18±0.03 0.16±0.01 0.14±0.01 0.16±0.01 0.17±0.01 0.16±0.01 0.16±0.01 0.26±0.03 0.21±0.03 0.15±0.02 Valine 1.72±0.2 1.61±0.22 1.61±0.22 1.75±0.17 1.45±0.12 1.31±0.12 1.64±0.15 1.09±0.1 1.04±0.1 1.59±0.1 1.55±0.1 1.32±0.11 Methionine 0.98±0.1 0.95±0.14 0.95±0.14 0.97±0.11 0.81±0.07 0.95±0.1 0.77±0.1 0.85±0.07 0.84±0.07 1.11±0.15 1.01±0.15 0.85±0.1 Isoleucine 2.48±0.22 2.35±0.31 2.35±0.31 2.20±0.15 2.08±0.2 2.07±0.21 2.19±0.2 2.02±0.17 1.95±0.17 2.41±0.3 2.33±0.3 2.20±0.19 Leucine 3.02±0.3 2.45±0.3 2.45±0.3 2.63±0.23 2.40±0.16 2.22±0.23 2.48±0.22 2.18±0.15 2.11±0.15 2.73±0.3 2.66±0.3 2.35±0.18 Tyrosine 2.06±0.2 1.87±0.2 1.85±0.2 1.42±0.14 1.67±0.1 1.52±0.13 1.74±0.12 1.55±0.1 1.53±0.1 1.98±0.2 1.94±0.2 1.74±0.1 Phenylalanine 1.72±0.2 1.88±0.22 1.86±0.22 1.17±0.11 1.48±0.1 1.31±0.12 1.53±0.1 1.39±0.11 1.36±0.11 1.73±0.2 1.66±0.2 1.54±0.1 Histidine 1.80±0.2 1.94±0.23 1.91±0.23 1.55±0.25 1.83±0.2 1.47±0.11 1.68±0.12 1.57±0.11 1.56±0.11 1.82±0.2 1.79±0.2 1.60±0.1 Lysine 1.12±0.1 0.99±0.17 0.95±0.17 0.77±0.09 0.85±0.1 0.66±0.02 0.78±0.06 0.80±0.08 0.78±0.08 0.92±0.1 0.87±0.1 0.81±0.1 Tryptophan 0.94±0.1 0.98±0.16 0.95±0.16 0.83±0.1 0.79±0.08 0.73±0.11 0.79±0.07 0.85±0.1 0.82±0.1 0.99±0.11 0.95±0.11 0.91±0.1 Arginine 1.10±0.1 1.11±0.16 1.01±0.16 1.15±0.13 0.78±0.09 0.82±0.12 0.75±0.1 0.78±0.1 0.75±0.1 0.83±0.09 0.79±0.09 0.69±0.05 Total 35.10±3.3 33.93±2.9 33.61±2.9 29.26±2.7 27.98±2.6 30.08±1.9 29.19±2.5 27.18±2.9 25.74±3.0 33.12±3.1 32.3±3.1 26.62±2.8

Averaged data of analyzed Cheese samples in triplicates.

during storage period, while the number of Enterococci) were quantitatively the dominant are a group of microorganisms that may influence Enterobacteriaceae remained high during the groups, and change of their viable numbers was the ripening process due to their proteolytic and ripening/storage period, despite a slight decrease significant (P≤0:01) throughout the ripening lipolytic activities and their ability to stimulate acid at the end of ripening period. All bacterial groups period. Numbers of Enterococcus sp. in all sam- production by some Lactococci (Sarantinopoulos except for coliforms were maximum in young ples of Ras cheese were almost the same in Blue et al., 2001). Total mesophilic aerobic bacteria cheeses. Numbers of Lactococcus sp. were cheese. The presence of Enterococci sp. in high increased reaching their highest numbers during a slightly higher than those of Lactobacilli and total numbers could be due to their tolerance to a wide 45 day ripening period at cold storage, and then mesophilic bacteria. The difference of Lactococci range of environmental conditions such as low rapidly declined. Numbers of microorganisms indi- counts from the other groups was maximum three temperature, high salt content and acidity cative of the hygienic quality, such as coliforms, log units. The predominance of Lactococcus sp. (Lorencová et al., 2012; Buňková et al., 2010). Enterococcus sp. and Lactococcus sp. were pre- during the early stages of raw milk cheeses ripe- Because of these properties, although all micro- sent in cheese at relatively high levels. These ning was reported (Manolopoulou et al., 2003). organisms were effected from salt significantly counts suggest that contamination was very high Lactic acid bacteria (Lactococci, Lactobacilli and (P≤0:05), Enterococcus sp. were not. Enterococci in raw milk. Numbers of coliforms and

El-Zahar 137

Table 4. Means counts of microorganisms in Egyptian market cheeses.

Ripening/ Coliform Moulds and Lactic acid storage Aerobic mesophilic Lactococci Enterococci Cheese type 6 group Yeasts 4 4 bacteria period bacteria (10 cfu/g) 2 3 (10 cfu/g) (10 cfu/g) 6 (month) (10 cfu/g) (10 cfu/g) (10 cfu/g) 6 7.59± 0.13b 4.98± .15ef 3.89 ± 0.21f 7.13± 0.19a 6.45± 0.22a 10.24±0.09a 12 7.37±0.42bc 5.07± .44de 4.05± 0.20ef 6.94± 0.31b 6.38± 0.87b 9.66±0.12b 24 6.84± 0.06fg 5.27± .59cde 4.33± 0.63de 6.75± 0.18c 6.33± .44bc 9.50±0.17bc Mish 36 6.28± 0.04i 5.34± .57cd 4.74± 0.90c 6.66± .06cde 6.13± 0.54c 9.23±0.08def 48 6.09± 0.18i 5.53± .35bc 4.82± 0.42c 6.34± 0.37f 5.88± 0.30d 9.15±0.13ef

6 7.13± 0.20de 4.5± 0.23g 4.05± 0.20ef 6.67±0.20cd 5.82±0.18de 9.65±0.20b

9 6.95± 0.24ef 4.63± 0.21g 4.37± 0.43d 6.58± .17de 5.78± .39de 9.55±0.13bc

12 6.65± 0.21gh 4.72± .42gf 4.79± 0.61c 6.50± .13ef 5.73± .47de 9.30±0.11de Ras 24 6.51± 0.18h 3.93± 0.23h 5.01± 0.39bc 6.40± 0.19f 5.63± 0.38e 9.13±0.07ef

6 7.92± 0.24a 5.84± 0.25a 4.82± 0.42c 4.51± 0.14g 5.88± 0.41d 9.36±0.011cd Blue-viened 9 7.37± 0.21bc 5.65± .26ab 5.23± 0.16ab 4.38± 0.21g 5.78± .44de 9.30±0.06de 12 7.22± 0.58ed 5.27± .19cde 5.37± 0.39a 4.00± 0.09h 5.73± .30de 9.08±0.08f

Microbiological composition of Egyptian market cheeses (means ± SD). Means log counts in triplicates.

Enterococcus sp. were not reduced significantly (P≤0:05), resulting of changing of pH and a decrease of acidity. As while numbers of Lactobacilli sp. were also reduced recommendation, the permissible level of biogenic significantly depending on the ripening time (P≤0:01), but amines stipulated by Egyptian Organization for Stan- they remained alive. This can be explained by the pH dardization and Quality Control (EOS, 1996) should be levels and the quantity of lactic acid. Counts of yeasts modified to meet the more safe standard adopted by and moulds were in Mish cheese similar to other findings Food Drug Administration (FDA, 2001) and their levels in Ras cheeses. During the ripening/storage period, the can be lowered by using good quality raw milk and numbers were not significantly decreased (P≤0:01), and maintaining hygiene standards during manufacturing and they had relatively high counts in Blue cheeses. Yeasts storage processes. were present at various levels among the distinct

cheeses, grouped from dairy markets, the differences in Conclusions and recommendations numbers may be due to the distinct pH and salt concen- trations found between the corresponding cheeses, The main feature influencing the BAs formation is the although no significant correlations resulted. Occurrence extent of growth of microorganisms, like Enterococci sp. of yeasts in cheeses was variable, because they have characterized by decarboxylase activity. The presence of been associated with the production of flavour com- high contents of BAs in Mish and Ras cheeses could be pounds as a result of their relatively strong proteolytic related to the enzymatic activity of proteases derived and lipolytic activities. However, scant information is from microorganisms, or from another factor, that is available regarding the contribution of yeasts to synthesis important from a qualitative point of view, that is, in of BAs in foods: a histidine-decarboxylase activity was relation to the type of amino acids provided to the amino found in yeasts of the genera Debaromyces and Candida acid decarboxylating microbiota, in particular tyrosine. isolated from fermented meat (Montel et al., 1999) and Therefore, a large amount of BAs in cheese reflects the such an activity was actually above that observed in lactic bad hygienic conditions under which they are produced acid bacteria. Macedo et al. (1995) found that the and stored. Accordingly, the levels of biogenic amines in presence of yeasts was closely related to lactic acid different cheeses should be in accordance with the safe utilization, while their contribution to the ripening process permissible limit recommended by FDA to ensure human was due to their proteolytic and lipolytic activities. In this safety. study, the number of microorganisms such as yeasts,

moulds and coliforms causing spoilage of cheeses by Conflict of Interests their putrefactive effects were decreased slightly. Forma- tion of basic compounds from proteolysis could be as a The author(s) have not declared any conflict of interests.

138 Afr. J. Food Sci.

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